LOCUS       sequence-343761-        5992 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HaloTag10 in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5992)
  AUTHORS   Frei MS, Tarnawski M, Roberti MJ, Koch B, Hiblot J, Johnsson K
  TITLE     Engineered HaloTag variants for fluorescence lifetime multiplexing.
  JOURNAL   Nat Methods. 2021 Dec 16. pii: 10.1038/s41592-021-01341-x. doi: 
            10.1038/s41592-021-01341-x.
  PUBMED    34916672
REFERENCE   2  (bases 1 to 5992)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2021 Dec 16. pii: 10.1038/s41592-021-01341-x. doi: 
            10.1038/s41592-021-01341-x."
COMMENT     Sequence Label: pET-51b-HaloTag10
FEATURES             Location/Qualifiers
     source          1..5992
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(87..106)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     297..318
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(570..1361,1362..1430))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1193..1212
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1431..1523)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1604..2192
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2093..2112
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2346..2363
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2378..2517
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2443..2462)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2603..2625
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2619..2810)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3382..4464)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     4426..4445
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4465..4542)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4748..4767
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4855..4874
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4855..4873
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4874..4898
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             4913..4935
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4945..4974
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             4975..4995
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(5894..5912)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5908..5955
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      join(5992,1..455)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
       61 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
      121 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
      181 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
      241 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
      301 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
      361 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
      421 cgcgaatttt aacaaaatat taacgtttac aatttctggc ggcacgatgg catgagatta
      481 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
      541 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
      601 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
      661 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
      721 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
      781 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
      841 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
      901 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
      961 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     1021 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     1081 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     1141 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     1201 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     1261 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     1321 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     1381 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     1441 tttcaatcat gattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     1501 atgtatttag aaaaataaac aaataggtca tgaccaaaat cccttaacgt gagttttcgt
     1561 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     1621 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     1681 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     1741 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     1801 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     1861 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     1921 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     1981 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     2041 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     2101 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     2161 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     2221 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     2281 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     2341 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     2401 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     2461 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     2521 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     2581 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     2641 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     2701 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     2761 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     2821 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     2881 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     2941 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     3001 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     3061 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     3121 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     3181 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     3241 gtcctcaacg acaggagcac gatcatgcta gtcatgcccc gcgcccaccg gaaggagctg
     3301 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     3361 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3421 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     3481 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     3541 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     3601 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     3661 gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     3721 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     3781 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     3841 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     3901 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     3961 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     4021 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     4081 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     4141 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     4201 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     4261 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     4321 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     4381 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     4441 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     4501 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     4561 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     4621 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     4681 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     4741 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     4801 cggccacgat gcgtccggcg tagaggatcg agatcgatct cgatcccgcg aaattaatac
     4861 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
     4921 actttaagaa ggagatatac catgcatcac caccatcatc accatcacca ccacgaaaac
     4981 ctgtattttc agggcatcgg tactggcttt ccattcgacc cccattatgt ggaagtcctg
     5041 ggcgagcgca tgcactacgt cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg
     5101 cacggtaacc cgacctcctc ctacgtgtgg cgcaacatca tcccgcatgt tgcaccgacc
     5161 catcgctgca ttgctccaga cctgatcggt atgggcaaat ccgacaaacc agacctgggt
     5221 tatttcttcg acgaccacgt ccgcttcatg gatgccttca tcgaagccct gggtctggaa
     5281 gaggtcgtcc tggtcattca cgactggggc tccgctctgg gtttccactg ggccaagcgc
     5341 aatccagagc gcgtcaaagg tattgcattt atggagttca tccgccctat cccgacctgg
     5401 gacgaatggc cagaatttgc ccgcgagacc ttccaggcct tccgcaccac cgacgtcggc
     5461 cgcaagctga tcatcgattg gaacgttttt atcgagggta cgctgccgat gggtgtcgtc
     5521 cgcccgctga ctgaagtcga gatggaccat taccgcgagc cgttcctgaa tcctgttgac
     5581 cgcgagccac tgtggcgctt cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc
     5641 gtcgcgctgg tcgaagaata catggactgg ctgcaccagt cccctgtccc gaagctgctg
     5701 ttctggggca ccccaggcgt tctgatccca ccggccgaag ccgctcgcct ggccaaaagc
     5761 ctgcctaact gcaaggctgt ggacatcggc ccgggtctga atctgctgca agaagacaac
     5821 ccggacctga tcggcagcga gatcgcgcgc tggctgtcta ctctggagat ttaattaacc
     5881 taggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     5941 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg ga
//
